DNA modified with C2\'-methoxy (C2\'-OMe) greatly enhances its resistance to nucleases, which is beneficial for the half-life of aptamers and DNA nanomaterials. Although the unnatural DNA polymerases capable of incorporating C2\'-OMe modified nucleoside monophosphates (C2\'-OMe-NMPs) were engineered via directed evolution, the detailed molecular mechanism by which an evolved DNA polymerase recognizes C2\'-OMe-NTPs remains poorly understood. Here, we present the crystal structures of the evolved Stoffel fragment of Taq DNA polymerase SFM4-3 processing the C2\'-OMe-GTP in different states. Our results reveal the structural basis for recognition of C2\'-methoxy by SFM4-3. Based on the analysis of other mutated residues in SFM4-3, a new Stoffel fragment variant with faster catalytic rate and stronger inhibitor-resistance was obtained. In addition, the capture of a novel pre-insertion co-existing with template 5\'-overhang stacking conformation provides insight into the catalytic mechanism of Taq DNA polymerase.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

The removal of mis-incorporated nucleotides by proofreading activity ensures DNA replication fidelity. Whereas the ε-exonuclease DnaQ is a well-established proofreader in the model organism Escherichia coli, it has been shown that proofreading in a majority of bacteria relies on the polymerase and histidinol phosphatase (PHP) domain of replicative polymerase, despite the presence of a DnaQ homolog that is structurally and functionally distinct from E. coli DnaQ. However, the biological functions of this type of noncanonical DnaQ remain unclear. Here, we provide independent evidence that noncanonical DnaQ functions as an additional proofreader for mycobacteria. Using the mutation accumulation assay in combination with whole-genome sequencing, we showed that depletion of DnaQ in Mycolicibacterium smegmatis leads to an increased mutation rate, resulting in AT-biased mutagenesis and increased insertions/deletions in the homopolymer tract. Our results showed that mycobacterial DnaQ binds to the β clamp and functions synergistically with the PHP domain proofreader to correct replication errors. Furthermore, the loss of dnaQ results in replication fork dysfunction, leading to attenuated growth and increased mutagenesis on subinhibitory fluoroquinolones potentially due to increased vulnerability to fork collapse. By analyzing the sequence polymorphism of dnaQ in clinical isolates of Mycobacterium tuberculosis (Mtb), we demonstrated that a naturally evolved DnaQ variant prevalent in Mtb lineage 4.3 may enable hypermutability and is associated with drug resistance. These results establish a coproofreading model and suggest a division of labor between DnaQ and PHP domain proofreader. This study also provides real-world evidence that a mutator-driven evolutionary pathway may exist during the adaptation of Mtb.

Werner syndrome (WS) is an autosomal recessive disease caused by loss of function of WRN. WS is a segmental progeroid disease and shows early onset or increased frequency of many characteristics of normal aging. WRN possesses helicase, annealing, strand exchange, and exonuclease activities and acts on a variety of DNA substrates, even complex replication and recombination intermediates. Here, we review the genetics, biochemistry, and probably physiological functions of the WRN protein. Although its precise role is unclear, evidence suggests WRN plays a role in pathways that respond to replication stress and maintain genome stability particularly in telomeric regions.

The measurement of dynamic changes in protein level and localization throughout the cell cycle is of major relevance to studies of cellular processes tightly coordinated with the cycle, such as replication, transcription, DNA repair, and checkpoint control. Currently available methods include biochemical assays of cells in bulk following synchronization, which determine protein levels with poor temporal and no spatial resolution. Taking advantage of genetic engineering and live-cell microscopy, we performed time-lapse imaging of cells expressing fluorescently tagged proteins under the control of their endogenous regulatory elements in order to follow their levels throughout the cell cycle. We effectively discern between cell cycle phases and S subphases based on fluorescence intensity and distribution of co-expressed proliferating cell nuclear antigen (PCNA)-mCherry. This allowed us to precisely determine and compare the levels and distribution of multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition complex subunit 1 (ORC1, and Claspin, with high spatiotemporal resolution in HeLa Kyoto cells. Combining these data with available mass spectrometry-based measurements of protein concentrations reveals the changes in the concentration of these proteins throughout the cell cycle. Our approach provides a practical basis for a detailed interrogation of protein dynamics in the context of the cell cycle.

During lagging strand chromatin replication, multiple Okazaki fragments (OFs) require processing and nucleosome assembly, but the mechanisms linking these processes remain unclear. Here, using transmission electron microscopy and rapid degradation of DNA ligase Cdc9, we observed flap structures accumulated on lagging strands, controlled by both Pol δ\'s strand displacement activity and Fen1\'s nuclease digestion. The distance between neighboring flap structures exhibits a regular pattern, indicative of matured OF length. While fen1Δ or enhanced strand displacement activities by polymerase δ (Pol δ; pol3exo-) minimally affect inter-flap distance, mutants affecting replication-coupled nucleosome assembly, such as cac1Δ and mcm2-3A, do significantly alter it. Deletion of Pol32, a subunit of DNA Pol δ, significantly increases this distance. Mechanistically, Pol32 binds to histone H3-H4 and is critical for nucleosome assembly on the lagging strand. Together, we propose that Pol32 establishes a connection between nucleosome assembly and the processing of OFs on lagging strands.

UNASSIGNED: Neddylation (NAE) inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report the cytotoxic vulnerability to NAE inhibitors in a subset of glioblastoma (GBM) preclinical models and identify genetic alterations and biological processes underlying differential response.
UNASSIGNED: GBM DNA sequencing and transcriptomic data were queried for genes associated with response to NAE inhibition; candidates were validated by molecular techniques. Multi-omics and functional assays revealed processes implicated in NAE inhibition response.
UNASSIGNED: Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and DNA repair pathways as significant differentiators between sensitive and resistant models. Vulnerability to MLN4924, a NAE inhibitor, is associated with elevated S-phase populations, DNA re-replication, and DNA damage. In a panel of GBM models, loss of WT PTEN is associated with resistance to different NAE inhibitors. A NAE inhibition response gene set could segregate the GBM cell lines that are most resistant to MLN4924.
UNASSIGNED: Loss of WT PTEN is associated with non-sensitivity to 3 different compounds that inhibit NAE in GBM. A NAE inhibition response gene set largely consisting of DNA replication genes could segregate GBM cell lines most resistant to NAEi and may be the basis for future development of NAE inhibition signatures of vulnerability and clinical trial enrollment within a precision medicine paradigm.

Homologous recombination (HR) plays an essential role in the repair of DNA double-strand breaks (DSBs), replication stress responses, and genome maintenance. However, unregulated HR during replication can impair genome duplication and compromise genome stability. The mechanisms underlying HR regulation during DNA replication are obscure. Here, we find that RTEL1 helicase, RAD51, and RAD51 paralogs are enriched at stalled replication sites. The absence of RTEL1 leads to an increase in the RAD51-mediated HR and fork reversal during replication and affects genome-wide replication, which can be rescued by co-depleting RAD51 and RAD51 paralogs. Interestingly, co-depletion of fork remodelers such as SMARCAL1/ZRANB3/HLTF/FBH1 and expression of HR-defective RAD51 mutants also rescues replication defects in RTEL1-deficient cells. The anti-recombinase function of RTEL1 during replication depends on its interaction with PCNA and helicase activity. Together, our data identify the role of RTEL1 helicase in restricting RAD51-mediated fork reversal and HR activity to facilitate error-free genome duplication.

Deleterious accumulation of R-loops, a DNA-RNA hybrid structure, contributes to genome instability. They are associated with BRCA1 mutation-related breast cancer, an estrogen receptor α negative (ERα-) tumor type originating from luminal progenitor cells. However, a presumed causality of R-loops in tumorigenesis has not been established in vivo. Here, we overexpress mouse Rnaseh1 (Rh1-OE) in vivo to remove accumulated R-loops in Brca1-deficient mouse mammary epithelium (BKO). R-loop removal exacerbates DNA replication stress in proliferating BKO mammary epithelial cells, with little effect on homology-directed repair of double-strand breaks following ionizing radiation. Compared to their BKO counterparts, BKO-Rh1-OE mammary glands contain fewer luminal progenitor cells but more mature luminal cells. Despite a similar incidence of spontaneous mammary tumors in BKO and BKO-Rh1-OE mice, a significant percentage of BKO-Rh1-OE tumors express ERα and progesterone receptor. Our results suggest that rather than directly elevating the overall tumor incidence, R-loops influence the mammary tumor subtype by shaping the cell of origin for Brca1 tumors.

Multipartite bacterial genomes pose challenges for genome engineering and the establishment of additional replicons. We simplified the tripartite genome structure (3.65 Mbp chromosome, 1.35 Mbp megaplasmid pSymA, 1.68 Mbp chromid pSymB) of the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Strains with bi- and monopartite genome configurations were generated by targeted replicon fusions. Our design preserved key genomic features such as replichore ratios, GC skew, KOPS, and coding sequence distribution. Under standard culture conditions, the growth rates of these strains and the wild type were nearly comparable, and the ability for symbiotic nitrogen fixation was maintained. Spatiotemporal replicon organization and segregation were maintained in the triple replicon fusion strain. Deletion of the replication initiator-encoding genes, including the oriVs of pSymA and pSymB from this strain, resulted in a monopartite genome with oriC as the sole origin of replication, a strongly unbalanced replichore ratio, slow growth, aberrant cellular localization of oriC, and deficiency in symbiosis. Suppressor mutation R436H in the cell cycle histidine kinase CckA and a 3.2 Mbp inversion, both individually, largely restored growth, but only the genomic rearrangement recovered the symbiotic capacity. These strains will facilitate the integration of secondary replicons in S. meliloti and thus be useful for genome engineering applications, such as generating hybrid genomes.